Report of a Sub-Committee of the 2011 FAO Consultation on Protein Quality Evaluation in Human Nutrition on:

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1 Report of a Sub-Committee of the 2011 FAO Consultation on Protein Quality Evaluation in Human Nutrition on: The assessment of amino acid digestibility in foods for humans and including a collation of published ileal amino acid digestibility data for human foods Members of the Sub-Committee 1 : Sarwar Gilani (Chair), Daniel Tomé, Paul Moughan and Barbara Burlingame (ex officio) 1 Dr Shane Rutherfurd of the Riddet Institute, Massey University, New Zealand assisted particularly with the collation of data on true ileal amino acid digestibility and was included as a co-author on the Sub-Committee s report. NOTE: the matters expressed in this report are those of the Sub-Committee and do not necessarily reflect the opinions of members (or a consensus) of the Expert Consultation The report was an integral part of the process, in achieving an overall consensus, as relayed in the overall report of the 2011 FAO Expert Consultation. First version written August A revised version (as presented here on website) was written and submitted to the R Uauy (Chair) Sub-Committee February The consensus statement from the Uauy Sub-Committee of April, 2012 (refer refers to the present revised report.

2 True ileal amino acid digestibility coefficients for application in the calculation of Digestible Indispensable Amino Acid Score (DIAAS) in human nutrition Paul J Moughan 1, Sarwar Gilani 2, Shane M Rutherfurd 1 and Daniel Tomé 3 1 Riddet Institute, Massey University, Private Bag , Palmerston North 4442, New Zealand 2 Nutrition Research Division, Health Canada, Ottawa, Ontario, Canada K1A OK9 3 AgroParisTech, INRA, UMR914 Nutrition Physiology and Ingestive Behavior, F Paris, France (revised report, February, 2012) Introduction Traditionally in human nutrition crude protein digestibility is assumed to accurately predict individual amino acid digestibility and is used in the calculation of the PDCAAS (protein digestibility corrected amino acid score). The digestibility of crude protein in foods has largely been determined on a faecal nitrogen digestibility basis (ie over the total digestive tract) in either human subjects or by using animal models (mainly the growing rat or growing pig). During the FAO Expert Consultation on Protein Quality Evaluation in Human Nutrition, held in Auckland, New Zealand, 31 March 2 April 2011, arguments were rehearsed that for accuracy, protein and amino acid digestibility in humans should be determined at the terminal ileum, as a measurement of amino acid disappearance between the mouth and the end of the small intestine. The Expert Consultation recommended specifically: 1. That proteins should firstly be described on the basis of their digestible amino acid contents, with each amino acid being treated as an individual nutrient; 2. that PDCAAS be replaced by a new score, DIAAS (digestible indispensable amino acid score) where DIAAS % = 100 x [(mg of digestible indispensable amino acid in 1 g of dietary protein)/(mg of the same indispensable amino acid in 1 g of reference protein)]; 3. in both cases the amounts of digestible dietary indispensable amino acids were to be determined based on amino acid composition and true ileal amino acid digestibility coefficients determined either in humans directly, the growing pig or the growing rat, in that order of preference. Although the physiological significance of the measurement of ileal amino acid digestibility was clearly recognised at the consultation, and indeed in earlier consultations (FAO/WHO, 1991; WHO/FAO/WHO, 2007) there were some practical concerns raised about the general availability of suitable ileal protein and amino acid digestibility data with application to humans. In this context an FAO Working Group was formed comprising Sarwar Gilani (Chair), Daniel Tomé, Paul Moughan and Barbara Burlingame (ex-officio), and the group was charged with developing a justification for the use of ileal protein and amino acid digestibility data in practice including: 1. demonstrating, based on experimental data, the nature of differences between protein digestibility and that of specific amino acids; 2. demonstrating, based on experimental data, the nature of ileo-faecal digestibility differences; 2

3 3. demonstrating that there currently exists a suitable quantum of ileal amino acid digestibility data, to allow its introduction for application in practice. These latter three objectives form the basis of this synopsis. 1) Basis for determining amino acid digestibility at the terminal ileum a. Faecal versus ileal digestibility a physiological perspective In simple-stomached animals possessing a well-developed hind-gut (and this includes humans), a profuse and diverse microbiota acts on undigested material entering the large bowel, with a significant degree of metabolism of protein, peptides and amino acids. Ammonia, one of the products of the bacterial breakdown of protein and amino acids, is absorbed from the hindgut, but amino acids, as such, are not considered to be absorbed from the large intestine in nutritionally meaningful amounts (Wrong et al., 1981; Moughan, 2003; Moughan and Stevens, 2012). Faecal protein is largely bacterial protein, and compositionally bears no resemblance to the array of dietary amino acids remaining undigested at the end of the ileum. Given that the bacterial protein does not directly relate to the food protein and undigested food amino acids, it is illogical to determine amino acid digestibility at the faecal level. Estimates of amino acid digestibility based on analysis of faeces do not describe the amounts of amino acid absorbed. Accordingly, measurements of digestibility determined at the ileal level are critical for determining amino acid losses of both dietary and endogenous origin (Moughan, 2003; Fuller and Tomé, 2005). Faecal-ileal digestibility differences can be substantial and both amino acid and protein ileo-faecal digestibility differences have been shown across a wide range of simple-stomached species of animal (Table 1). There is no reason to believe that the human, with a well-developed colon, would be any different, and indeed albeit limited experimental evidence with humans supports this. It should be noted that ileal values of amino acid digestibility may themselves not be completely accurate estimators of amino acid uptake as there may be unaccounted for microbial catabolism and synthesis of amino acids in the upper digestive tract. Fuller (2012) has discussed recent experimental findings on bacterial amino acid synthesis in the upper gastro-intestinal tract, where absorption of the synthesised amino acid may occur. He concludes, that although there are still uncertainties about the impact of microbial activity in the upper digestive tract, the amino acid composition of ileal digesta provides the best available basis for estimating amino acid digestibility. Also several carefully controlled studies with simple-stomached animals have demonstrated the accuracy of ileal amino acid digestibility values (refer later section). b. Crude protein versus amino acid digestibility With the PDCAAS method a single value for the digestibility of crude protein is used to adjust dietary concentrations of dietary indispensable amino acids. The digestibility of crude protein is assumed to apply to individual dietary indispensable amino acids and this assumes that differences between protein digestibility and amino acid digestibility are minor. This is not the case. Significant differences can be observed between protein digestibility and the digestibility of specific amino acids and among amino acid digestibilities. Such differences are highlighted in the data selected from rat studies and shown in Table 2, which were reported in the FAO/WHO (1991) report on the joint FAO/WHO expert consultation on protein quality evaluation held in 1989, and are sourced from work from Sarwar Gilani s 3

4 laboratory (Sarwar Gilani, 1987). Maximum and minimum true ileal amino acid digestibility values determined in pigs along with true ileal protein digestibility for a wider range of foods are shown in Figure 1. Clearly there can be practically significant differences between crude protein digestibility and that of specific amino acids. Amino acid digestibility should be used in estimating dietary protein quality wherever possible. c. Endogenous amino acids present in terminal ileal digesta During the digestion of food very considerable quantities of proteins of body (endogenous protein) as opposed to dietary origin are voided into the digestive tract. Much of this material is recycled, with the protein being digested and the amino acids reabsorbed. Nevertheless large quantities of endogenous protein, peptides and amino acids remain unabsorbed at the end of the small intestine and these along with endogenous protein originating from the colon are largely catabolised by the colonic microflora, and (for the dietary indispensable amino acids) represent a loss of amino acids from the body. If dietary amino acid digestibility is to be determined at the terminal ileum, and given that the ileal digesta contain copious quantities of endogenous proteins, it becomes necessary to determine the endogenous amino acid component. If coefficients of amino acid digestibility are not corrected for the ileal endogenous amino acids, the resultant digestibility coefficients are referred to as apparent coefficients, whereas if the correction is made the coefficients are termed true. True digestibility is a fundamental property of the food and is not affected by the dietary conditions under which the food is given to the subject. The apparent digestibility measure will be affected by the assay conditions and is, therefore, variable and open to error. At a set food dry-matter intake, whereby the ileal endogenous flow may be constant, the determined apparent amino acid digestibility coefficient increases markedly and curvilinearly from low to higher dietary amino acid contents. This is an artefact of the assay, and reflects a disproportionate influence of the uncorrected for ileal endogenous amino acid flow, at the lower amino acid intakes. This effect is shown clearly by the experimental data of Donkoh and Moughan (1994) (Figure 2) in which semi-synthetic corn-starch based diets containing different amounts of meat-and-bone meal protein were fed to growing rats and ileal digesta collected from the euthanased animals. Apparent ileal N digestibility increased with increasing dietary protein content, from a low of 65% to a high of 75%, whereas true ileal N digestibility was around 77% and independent of the dietary protein content. Clearly, determined ileal amino acid flows need to be corrected for the ileal endogenous amino acids. Traditionally, this has been done by feeding the human subject or animal (model for human) a protein-free diet, but this method has been criticised as being unphysiological (Low, 1980). Other more physiological methods (eg the enzyme hydrolysed protein/ultrafiltration method; stable isotope-labelled protein) have been developed (Moughan et al., 1998; Bos et al, 2002). The practical application of these methods to give true or standardised estimates of ileal protein and amino acid digestibility has been the subject of review (Fouillet et al, 2002; Fuller and Tomé, 2005; Columbus and de Lange, 2012; Moughan and Rutherfurd, 2012). 2) Faecal and ileal nitrogen and amino acid digestibility in the pig An important body of comparative ileo-faecal N and amino acid digestibility data is found in work with the growing pig (Low, 1980), which appears to be a suitable animal model for nutrient digestibility studies in humans, particularly for the determination of ileal nitrogen digestibility (Pond and Houpt, 1978; Miller and Ullrey, 1987; Moughan and Rowan, 1989; Moughan et al., 1992; Moughan, et al., 1994; Deglaire et al., 2009; Deglaire and Moughan, 2012). 4

5 a. Comparisons of ileo-faecal nitrogen and amino acid digestibility in the pig There is general agreement across studies, that the ileal digestibilities of most amino acids are lower than corresponding digestibilities determined over the total digestive tract (for example see Table 3), but this finding is not universal. The amount of amino acids disappearing in the large intestine usually ranges from around 5% to 35% of the amino acid ingested. It appears that the lower the overall ileal digestibility of nitrogen or amino acids, the greater is the ileofaecal difference in digestibility (Table 3). This is understandable as with diets containing highly digestible protein most is absorbed before the digesta enter the large intestine, whereas with protein sources of lower quality, there are larger residues to be fermented and with a proportionately greater disappearance of amino acids between the terminal ileum and rectum. The extent of faecal digestibility over- or under-estimation varies with the amino acid, the type of dietary protein and the influence of other dietary components. Lenis (1983) surveyed the world literature from 1964 to 1982 for some 35 foods. For threonine and tryptophan the mean overestimations of apparent digestibility by the faecal method in comparison with ileal, were 10 and 11 percentage units, respectively. The ileo-faecal differences tended to be smaller for lysine. The faecal method overestimated (mean overestimation = 5.6% units) lysine digestibility for eleven foods and underestimated it (mean underestimation = 4.3% units) in ten further foods. Faecal values appear to often considerably underestimate the actual (ileal) digestibility of methionine, although the opposite has been found for cysteine. Hendriks et al., (2012) have collated apparent faecal and ileal N digestibility data for the growing pig from a large number of studies. Generally faecal digestibility values were much higher than ileal digestibility values, but in a few cases the ileal N digestibility value exceeded its faecal counterpart. The extensive data set also clearly demonstrates that as apparent ileal N digestibility increased from a low of 50% to a high of 95%, the ileo-faecal difference decreased quite markedly. Overall, the published evidence suggests that in the growing pig, ileal amino acid digestibility values are quantitatively different from faecal amino acid digestibility values, and ileal values are superior for application in practice. Similar ileo-faecal protein digestibility differences have been reported in the growing rat (Table 4). b. Experimental evidence for the validity of ileal amino acid digestibility coefficients The effect of hindgut microbial metabolism of undigested dietary amino acids, on faecal estimates of digestibility may explain the frequently reported low statistical correlations observed between pig growth performance and faecal estimates of amino acid uptake (Crampton and Bell, 1946; Lawrence, 1967; Cole et al., 1970). Whereas faecal amino acid digestibility coefficients have been poor predictors of animal growth, ileal amino acid digestibility values have been shown to be sensitive in detecting small differences in protein digestibility due to the processing of foods (van Weerden et al., 1985; Sauer and Ozimek, 1986) and several studies (Tanksley and Knabe, 1980; Low et al., 1982; Just et al., 1985; Moughan and Smith, 1985; Dierick et al., 1988) have demonstrated that ileal values are accurate in describing the extent of uptake of amino acids from the gut lumen. Rutherfurd et al (1997) undertook a study to evaluate the accuracy of true ileal lysine digestibility coefficients in predicting whole body lysine deposition in the pig. The study supported the accuracy of true ileal lysine digestibility as a predictor of dietary lysine uptake from the digestive tract. Experimental evidence for the validity of the application of ileal amino acid digestibility coefficients has recently been reviewed by Columbus and de Lange (2012) who 5

6 conclude that there is a large body of evidence to suggest that in many instances measures of ileal amino acid digestibility yield reasonable estimates of bioavailability of amino acids in foods. c. The digestibility of lysine from processed foods For foods that have been subjected to processing and with possible damage to amino acids, the traditional ileal digestibility assay is not expected to accurately indicate the absorption of all amino acids (Moughan et al., 1991). This is well exemplified for lysine. During amino acid analysis with strong hydrochloric acid, early Maillard compounds are known to partially revert to lysine. Such reversion does not occur, however, in the alimentary canal during gastric digestion. Consequently, estimates of dietary and ileal digesta lysine will be in error leading to biased ileal digestibility coefficients. Although, and at least for lysine, structurally unaltered molecules can be accurately determined chemically (eg FDNB lysine assay), there is evidence (Hurrell and Carpenter, 1981) that the unaltered or chemically available molecules may not be fully absorbed from the damaged proteins. The absorption (measured at terminal ileum) of reactive lysine has been determined in a study (Moughan et al., 1996) with the growing pig (Table 5). A casein-glucose mixture was heated to produce early Maillard compounds, and the amount of epsilon-n-deoxy-fructosyl-lysine (blocked lysine) and lysine regenerated after acid hydrolysis in the resulting material was calculated from the determined level of furosine. The amount of unaltered or reactive lysine was found by difference between the total lysine (acid hydrolysis) and regenerated lysine. The FDNB method allowed accurate assessment of the amount of chemically reactive lysine, which was grossly overestimated by conventional amino acid analysis (acid-hydrolysed lysine), but the reactive lysine was not completely absorbed. For the amino acid lysine, and in foods that may have sustained chemical alteration of their proteins, reactive lysine as opposed to total lysine (traditional amino acid analysis) should be determined on both the food and ileal digesta, and should be used in the calculation of digestibility (Moughan and Rutherfurd, 1996; Rutherfurd and Moughan 2012). True ileal digestible reactive lysine provides an accurate assessment of lysine available to the tissues for metabolism. 3) Ileal protein and amino acid digestibility in the adult human a. Nitrogen flow at the terminal ileum in humans In the adult human, total nitrogen flow at the terminal ileum ranges from 2 to 5 g/day, with endogenous and dietary nitrogen losses ranging from 0.7 to 4 g/day and 0.3 to 1 g/day, respectively. Endogenous and dietary amino acid losses are g/day and g/day, respectively (Chacko and Cummings, 1988; Mahé et al., 1992; Rowan et al., 1993; Fuller et al., 1994 ; Gausserès et al., 1996; Mariotti et al., 1999; Gaudichon et al., 2002; Moughan et al., 2005a). These results show that a significant proportion of the nitrogen flow (about 40% to 50%) in the human ileum is of non-protein origin. b. Ileal versus faecal nitrogen digestibility in humans Sammons (1961) determined daily rates of faecal N output from normal human subjects and ileal N output from ileostomates given the same diet. The ileal output was 2.7 g N/d and the faecal output 1.8 g N/d, demonstrating a considerable loss of N in the large bowel of the human and suggesting quantitatively important differences in ileal and faecal N digestibility. Sandstrom et al (1986) gave soya- and meat-based diets to ileostomates and reported 6

7 apparent ileal digestibility coefficients for total N in the range of 80 to 85%. In comparison, in human subjects receiving soya-based diets, true faecal digestibility coefficients ranging from 90 to 98% have been reported (Istfan et al., 1983; Scrimshaw et al., 1983; Wayler et al., 1983; Young et al., 1984). Evenepoel et al (1998) fed 15 N-labelled egg protein to human ileostomates and recorded true ileal digestibility values for crude protein in cooked and raw egg of 90.0 and 51.3%, respectively and concluded that the ileal digestibility value for cooked egg was lower than the comparable published range for faecal digestibility (92-97%). In contrast, Gibson et al (1976) reported only marginally lower digestibility coefficients determined at the terminal ileum rather than across the total digestive tract for human subjects receiving highly digestible proteins. Bos et al (1999) measured true ileal and faecal protein digestibility using 15N-labelled milk protein and showed that the amount of N recovered at the terminal ileum peaked after 1 h and then decreased during the next 7 h with no significant amount of exogenous N recovered at the terminal ileum at the end of the 8 h, whereas the amount recovered in the faeces remained at a very low level after 24 h, peaked after 60 h and progressively decreased (figure 3). The true ileal and faecal digestibilities of the highly digestible milk protein were calculated as 95.5 % and 96.6% respectively, and were not statistically significantly different from each other. These results from human digestibility studies, albeit small in number, are in general agreement with observations in other simple-stomached mammals. c. Ileal versus faecal amino acid digestibility in humans and ileal amino acid digestibility values for humans In the study of Rowan et al (1994) five subjects with established ileostomies and six normal subjects consumed a constant diet consisting of meat, vegetables, fruit, bread and dairy products for 7 d with collection of ileostomy contents or faeces, respectively, over the final 4 d of the experimental period. Generally the apparent faecal digestibility coefficients were higher than their ileal counterparts with significant (P < 0.05) differences being recorded for arginine, aspartic acid, glycine, phenylalanine, proline, serine, threonine and tryptophan (Table 5). The faecal digestibility of methionine was statistically significantly lower than the ileal value. Some of the differences recorded were quantitatively important (eg methionine and tryptophan), and particularly when viewed against the background of the ileal values being determined using ileostomates. Ileostomates develop a characteristic and quite extensive microflora at the end of the ileum (Vince et al., 1973). It is concluded that the use of faecal amino acid digestibility coefficients may be misleading for determining the uptake of dietary amino acids in humans, and ileal digestibility coefficients are preferred for application in humans. Results for a set of studies determining true ileal nitrogen digestibility in healthy humans, using stable isotope-labelled protein, are reported in table 7. The values for true ileal digestibility ranged from 84 to 95%. Apparent and true ileal amino acid digestibility for mixed meals based on intact casein or on hydrolyzed casein were determined in healthy adult humans and showed differences among amino acids (Deglaire et al, 2009) (Table 8). True ileal amino acid digestibility for milk and soya protein was also determined in healthy human subjects (Gaudichon et al, 2002). The lowest digestibility was observed for threonine in the soya group (89.0%) and the highest was for tyrosine in the milk group (99.3%) (Table 9). A significantly lower digestibility was found for threonine, valine, histidine, tyrosine, alanine, and proline with soya protein as compared with milk protein. Nitrogen digestibility was 7

8 significantly lower in the soya group than in the milk group. In contrast, when total nitrogen digestibility was calculated from individual amino acid digestibilities, the difference between milk and soya was not statistically significant. 4) General considerations a. Findings of the 2011 FAO Expert Consultation The digestible indispensable amino acid score (DIAAS) approach is recommended for the evaluation of dietary protein quality in humans. In the calculation of DIAAS a value for the amount of digestible indispensable amino acid is used. With respect to the determination of dietary digestible indispensable amino acid content the 2011 FAO Consultation concluded: It is recommended that protein quality assessment should be based on the true ileal digestibility values of individual amino acids rather than overall (faecal) digestibility. True ileal amino acid digestibility should preferably be determined in humans. Where human data are lacking, it is recommended that ileal amino acid digestibility values from the growing pig be used, and where these data are not available, from the growing rat. When amino acid digestibility data are not available, amino acid digestibility is assumed to be equivalent to crude protein digestibility. In this case true ileal crude protein digestibility data are preferable, but where unavailable true faecal crude protein digestibility may be used. b. Published data on true ileal amino acid digestibility in foods for humans A review of the literature and various data bases has been undertaken and true ileal digestibility data are presented (refer attached appendix) from work with adult humans, the growing pig and the growing laboratory rat. The dataset once again demonstrates a considerable degree of variation in digestibility among foods, and among individual amino acids and total nitrogen within a food, highlighting the need to use digestibility data for individual amino acids and specific foods wherever possible. The true ileal amino acid digestibility database presented herewith, has been gleaned from a large number of diverse studies conducted over a number of years and where different methodologies have been used. Although each study has been assessed to ensure bona-fide approaches were employed, nevertheless considerable methodological-based variation will be inherent. Also, in many cases only a rudimentary description of the food source is available. Thus the present dataset should be regarded as interim. There is a need to develop studies and to accumulate data on ileal amino acid digestibility directly determined in humans. In addition, a validated and standardised method should be developed using animal models (either the growing pig or the growing rat). Several currently used methodologies for obtaining pig or rat true ileal digestibility data could be considered acceptable, but the development of an agreed standardised methodology (see Moughan and Rutherfurd, 2012) would be a considerable advancement. Such an assay would be applied to form a comprehensive standardised set of tables for the true ileal amino acid digestibility of human foods. 8

9 Table 1. Comparison of ileal and faecal digestibility of dietary protein for the domestic chicken and several simple-stomached mammals Apparent digestibility (%) Difference (faecalileal, Ileal Faecal % units) Piglet % Growing pig % Pre-ruminant calf % Chicken % Growing rat % Growing rat % Growing rat % Growing rat % 1 Piglets (6 kg liveweight) fed bovine milk (Moughan et al., 1990) 2 Pig (45 kg liveweight) given meat and bone meal based diet (Moughan et al., 1984) 3 Milk fed calf (45 kg liveweight) (Moughan et al., 1989) 4 Overall mean amino acid digestibility for 9 amino acids and 16 diets given to 10 week old chickens (Raharjo and Farrell 1984) and based on a collection of ileal digesta or excreta. Rat (80 g bodyweight) given a diet based either on meat and bone meal 5, fish meal 6, field peas meal 7 or barley meal 8 (Moughan et al., 1984) 9

10 Table 2. Faecal crude protein and amino acid digestibility (%) for selected foods, determined in the growing rat Protein Lys Met Cys Thr Trp Pea flour Pea (autoclaved) Pintobean (canned) Lentil (autoclaved) Fababean (autoclaved) Soyabean Peanut Wheat from Sarwar Gilani (1987) 10

11 Table 3. Apparent ileal and faecal digestibilities (%) of dietary amino acids in the growing pig a) Pigs fed a balanced cereal-based diet (Sauer and Just, 1979, n = 30). Ileum Faeces Difference (faecal-ileal, % units) Arginine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine Average b) Pigs fed wheat flour and wheat offal with digestibility determined at the terminal ileum (I) and in faeces (F) (Sauer et al., 1977). Amino Acid Wheat flour Wheat offal I F I F Lysine Histidine Methionine Isoleucine Leucine c) Pigs fed raw soyabean flour (Nutrisoy) and autoclaved Nutrisoy. 1 Nutrisoy Autoclaved Nutrisoy Ileal Faecal Ileal Faecal Protein Arginine Histidine Isoleucine Leucine Lysine Methionine Cysteine Phyenylalanine Tyrosine Threonine Valine Abstracted from Li et al. (1998); Two maize starch-based diets containing 200 g/kg diet of either Nutrisoy (a defatted soya flour containing active trypsin inhibitors) or autoclaved Nutrisoy (containing reduced amounts of trypsin inhibitors) were tested. 2 Digestibility after correction for dietary supplementation of methionine. 11

12 Table 4. Mean apparent digestibility of crude protein (%) as determined in ileal digesta or faeces in the growing rat (Moughan et al., 1984) Barley meal Meat and bone Fish meal Field peas meal Ileal Faecal Difference (faecal-ileal, % units) +1% +9% +6% -4% 12

13 Table 5. Amounts of acid-hydrolysed lysine, FDNB lysine, reactive lysine and absorbed reactive lysine in a heated casein-glucose mixture. Acidhydrolysed FDNB 2 Reactive 3 Absorbed 1 reactive 4 Lysine (g 100 g -1 ) After conventional amino acid analysis. 2 FDNB = fluoro-dinitrobenzene. 3 Lysine units remaining chemically reactive after heating, determined from furosine levels. 4 Reactive lysine absorbed by the end of the small intestine. From Moughan et al. (1996). 13

14 Table 6: Mean apparent ileal and faecal amino acid digestibility coefficients for adult ileostomate and healthy human subjects (65 kg body weight) receiving a meat, vegetable, cereal, and dairy-product-based diet, respectively (from Rowan et al., 1994) Digestibility coefficient 1 Amino acid Ileal (n 5) Faecal (n 6) Statistical significance Difference (Faecal-ileal, % units) Arginine * +3 Aspartate * +3 Serine *** +5 Threonine ** +4 Proline ** +5 Glycine *** +15 Phenylalanine *** +1 Methionine *** -10 Tryptophan * +6 1 Amino acids for which significant (P<0.05) ileo-faecal differences were found. 14

15 Table 7: Ileal nitrogen digestibility determined in humans Protein Ileal digestibility Reference Apparent True Milk protein Mahé et al, 1994 ; Bos et al, 2003; Gaudichon et al, 2002 Fermented milk 90 - Mahé et al, 1994 Casein Deglaire et al, 2009 Soya protein Bos et al, 2003; Gaudichon et al, 2002 Pea protein Gausserès et al, Gausserès et al, Mariotti et al, 2001 Wheat Bos et al, Juillet et al, 2008 Lupin protein Mariotti et al, 2002 Rapeseed protein - 84 Bos et al,

16 Table 8: Apparent and true ileal amino acid digestibility for mixed meals based on intact casein or on hydrolysed casein fed to adult humans (Mean values and pooled standard deviations) (Deglaire et al., 2009) Intact casein Hydrolysed casein Apparent True Apparent True Indispensable amino acids Histidine Isoleucine Leucine Lysine Phenylalanine Threonine Tyrosine Valine Dispensable amino acids Alanine Aspartic acid Glutamic acid Proline Serine

17 Table 9: True ileal digestibility (%) of dietary nitrogen and amino acids for milk or soya Protein in healthy human volunteers (from Gaudichon et al, 2002) Milk Soya Aspartate + asparagine 94.3 ± ± 4.0 Serine 92.0 ± ± 3.9 Glutamate + glutamine 95.3 ± ± 2.8 Proline 96.1 ± 2.2 a 92.8 ± 3.8 Glycine 91.6 ± ± 5.1 Alanine 95.9 ± 1.9 a 92.3 ± 2.5 Tyrosine 99.3 ± 0.4 a 96.8 ± 1.5 Threonine 93.4 ± 2.3 a 89.0 ± 4.9 Valine 95.9 ± 1.9 a 92.5 ± 3.5 Isoleucine 95.4 ± ± 3.1 Leucine 95.1 ± ± 3.0 Phenylalanine 95.6 ± ± 2.3 Lysine 94.9 ± ± 2.5 Histidine 94.9 ± 2.7 a 91.7 ± 1.7 Average amino acid digestibility b 95.3 ± ± 3.0 Nitrogen digestibility 95.3 ± 0.9 a 91.7 ± 1.8 NOTE. Values are means ± SD, (n = 7 and n = 6 for milk and soya, respectively). a Significantly different from soya group (ANOVA, P <0.05). b Calculated from amino acid digestibilities weighted by the proportion of each amino acid in the dietary protein. 17

18 Figure 1. Maximum true ileal amino acid digestibility ( ), minimum true ileal amino acid digestibility ( ), and true ileal nitrogen digestibility ( ). Source of data: AFZ, Ajinomoto Eurolysine, Aventis Animal Nutrition, INRA, ITCF (2000); Han et al. (2006) and Moughan et al. (2005b). All values are from studies with the growing pig except for the last three datasets (whey protein isolate, soya protein isolate, soya protein concentrate) which were obtained with obtained with adult humans. 18

19 Figure 2. Effect of dietary protein content on mean apparent ( ) and true (+) ileal N digestibility values for rats given a meat-and-bone-meal-based diet (Donkoh and Moughan, 1994). 19

20 Figure 3: Exogenous nitrogen recovered in the ileum (o) and in the faeces ( ) following ingestion of [15N]milk by healthy adults after an overnight fast. Each value represents the mean of five subjects (Bos et al, 1999). 20

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